Promoters for expressing genes in a fungal cell

ABSTRACT

The present invention relates to isolated promoters and constructs, vectors, and fungal host cells comprising such promoters operably linked to polynucleotides encoding polypeptides. The present invention also relates to methods for producing such polypeptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 13/885,145, filed Dec. 15, 2011, now U.S. Pat. No. 9,284,588,which is a National Phase filing under 35 U.S.C. §371 of InternationalApplication No. PCT/US2011/065287, filed Dec. 15, 2011, which claimspriority to U.S. Provisional Application Ser. No. 61/423,909, filed Dec.16, 2010. The contents of these applications are hereby incorporated byreference in their entireties.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form.The computer readable form is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to methods for producing polypeptides. Thepresent invention also relates to isolated promoters and to nucleic acidconstructs, vectors, and host cells comprising the promoters operablylinked to polynucleotides encoding the polypeptides.

Description of the Related Art

The recombinant production of a polypeptide in a fungal host cell, e.g.,a filamentous fungal cell, may provide for a more desirable vehicle forproducing the polypeptide in commercially relevant quantities.

Recombinant production of a polypeptide is accomplished by constructingan expression cassette in which the DNA coding for the polypeptide isplaced under the expression control of a promoter, excised from a gene,suitable for the host cell. The expression cassette is introduced intothe host cell, usually by plasmid-mediated transformation. Production ofthe polypeptide is then achieved by culturing the transformed host cellunder inducing conditions necessary for the proper functioning of thepromoter contained on the expression cassette.

The use of a fungal host cell for the recombinant production ofpolypeptides generally requires the availability of promoters that aresuitable for controlling the expression of the polypeptides in the hostcell. Consequently, there is a need in the art for new promoters forcontrolling the recombinant expression of genes.

The present invention provides improved methods for producing apolypeptide in a fungal host cell.

SUMMARY OF THE INVENTION

The present invention relates to methods for producing a polypeptide,comprising: (a) cultivating a fungal host cell in a medium conducive forthe production of the polypeptide, wherein the fungal host cellcomprises a polynucleotide encoding the polypeptide operably linked to apromoter selected from the group consisting of (i) a promoter comprisinga nucleotide sequence having at least 60% sequence identity to SEQ IDNO: 1; (ii) a promoter comprising a nucleotide sequence that hybridizesunder at least medium stringency conditions with SEQ ID NO: 1 or thefull-length complement thereof; (iii) a promoter comprising SEQ ID NO:1; (iv) a promoter comprising a subsequence of (i), (ii), or (iii) thatretains promoter activity; and (v) a mutant, hybrid, or tandem promoterof (i), (ii), (iii), or (iv); wherein the polynucleotide encoding thepolypeptide is foreign to the promoter; and (b) isolating thepolypeptide from the cultivation medium.

The present invention also relates to isolated promoters selected fromthe group consisting of (i) a promoter comprising a nucleotide sequencehaving at least 60% sequence identity to SEQ ID NO: 1; (ii) a promotercomprising a nucleotide sequence that hybridizes under at least mediumstringency conditions with SEQ ID NO: 1 or the full-length complementthereof; (iii) a promoter comprising SEQ ID NO: 1; (iv) a promotercomprising a subsequence of (i), (ii), or (iii) that retains promoteractivity; and (v) a mutant, hybrid, or tandem promoter of (i), (ii),(iii), or (iv).

The present invention also relates to constructs, vectors, and fungalhost cells comprising a promoter of the present invention operablylinked to a polynucleotide encoding a polypeptide.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a restriction map of pSMai226.

FIG. 2 shows a restriction map of pSaMe-CatP.

FIG. 3 shows the DNA sequence of a Trichoderma reesei catalase promoter(SEQ ID NO: 1).

DEFINITIONS

Allelic variant: The term “allelic variant” means any of two or more(e.g., several) alternative forms of a gene occupying the samechromosomal locus. Allelic variation arises naturally through mutation,and may result in polymorphism within populations. Gene mutations can besilent (no change in the encoded polypeptide) or may encode polypeptideshaving altered amino acid sequences. An allelic variant of a polypeptideis a polypeptide encoded by an allelic variant of a gene.

cDNA: The term “cDNA” means a DNA molecule that can be prepared byreverse transcription from a mature, spliced, mRNA molecule obtainedfrom a eukaryotic or prokaryotic cell. cDNA lacks intron sequences thatmay be present in the corresponding genomic DNA. The initial, primaryRNA transcript is a precursor to mRNA that is processed through a seriesof steps, including splicing, before appearing as mature spliced mRNA.

Coding sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of a polypeptide. Theboundaries of the coding sequence are generally determined by an openreading frame, which begins with a start codon such as ATG, GTG, or TTGand ends with a stop codon such as TAA, TAG, or TGA. The coding sequencemay be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

Control sequences: The term “control sequences” means nucleic acidsequences necessary for expression of a polynucleotide encoding apolypeptide. Each control sequence may be native (i.e., from the samegene) or foreign (i.e., from a different gene) to the polynucleotideencoding the polypeptide. Such control sequences include, but are notlimited to, a leader, polyadenylation sequence, propeptide sequence,promoter, signal peptide sequence, and transcription terminator. At aminimum, the control sequences include a promoter, and transcriptionaland translational stop signals. The control sequences may be providedwith linkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe polynucleotide encoding the polypeptide.

Expression: The term “expression” includes any step involved in theproduction of a polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding apolypeptide and is operably linked to control sequences that provide forits expression.

High stringency conditions: The term “high stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 50% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at65° C.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, or the like, with anucleic acid construct or expression vector comprising a polynucleotideof interest. The term “host cell” encompasses any progeny of a parentcell that is not identical to the parent cell due to mutations thatoccur during replication.

Hybrid promoter: The term “hybrid promoter” means portions of two ormore (e.g., several) promoters that are linked together to generate asequence that is a fusion of the portions of the two or more promoters,which when operably linked to a coding sequence mediates thetranscription of the coding sequence into mRNA.

Isolated: The term “isolated” means a substance in a form or environmentthat does not occur in nature. Non-limiting examples of isolatedsubstances include (1) any non-naturally occurring substance, (2) anysubstance including, but not limited to, any enzyme, variant,polynucleotide, protein, peptide or cofactor, that is at least partiallyremoved from one or more or all of the naturally occurring constituentswith which it is associated in nature; (3) any substance modified by thehand of man relative to that substance found in nature; or (4) anysubstance modified by increasing the amount of the substance relative toother components with which it is naturally associated (e.g., multiplecopies of a gene encoding the substance; use of a stronger promoter thanthe promoter naturally associated with the gene encoding the substance).A polypeptide of interest may be used in industrial applications in theform of a fermentation broth product, that is, the polypeptide is acomponent of a fermentation broth used as a product in industrialapplications (e.g., ethanol production). The fermentation broth productwill in addition to the polypeptide of interest comprise additionalingredients used in the fermentation process, such as, for example,cells (including, the host cells containing the gene encoding thepolypeptide of interest which are used to produce the polypeptide), celldebris, biomass, fermentation media and/or fermentation products. Thefermentation broth may be optionally subjected to one or morepurification (including filtration) steps to remove or reduce one morecomponents of a fermentation process. Accordingly, an isolated substancemay be present in such a fermentation broth product.

Low stringency conditions: The term “low stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 25% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at50° C.

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc. It is known in the art that a hostcell may produce a mixture of two of more different mature polypeptides(i.e., with a different C-terminal and/or N-terminal amino acid)expressed by the same polynucleotide.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” means a polynucleotide that encodes a mature polypeptidehaving biological activity.

Medium stringency conditions: The term “medium stringency conditions”means for probes of at least 100 nucleotides in length, prehybridizationand hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/mlsheared and denatured salmon sperm DNA, and 35% formamide, followingstandard Southern blotting procedures for 12 to 24 hours. The carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS at 55° C.

Medium-high stringency conditions: The term “medium-high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 60° C.

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs expression of the coding sequence.

Polypeptide fragment: The term “polypeptide fragment” means apolypeptide having one or more (e.g., several) amino acids absent fromthe amino and/or carboxyl terminus of a mature polypeptide; wherein thefragment has biological activity. In one aspect, the fragment has atleast 85%, e.g., at least 90% or at least 95% of the number of aminoacids as the mature polypeptide.

Polypeptide variant: The term “polypeptide variant” means a polypeptidehaving biological activity comprising an alteration, i.e., asubstitution, insertion, and/or deletion, at one or more (e.g., several)positions. A substitution means replacement of the amino acid occupyinga position with a different amino acid; a deletion means removal of theamino acid occupying a position; and an insertion means adding an aminoacid adjacent to and immediately following the amino acid occupying aposition.

Promoter: The term “promoter” means a DNA sequence that binds RNApolymerase and directs the polymerase to the correct downstreamtranscriptional start site of a polynucleotide encoding a polypeptide toinitiate transcription. RNA polymerase effectively catalyzes theassembly of messenger RNA complementary to the appropriate DNA strand ofthe coding region. The term “promoter” will also be understood toinclude the 5′ non-coding region (between promoter and translationstart) for translation after transcription into mRNA, cis-actingtranscription control elements such as enhancers, and other nucleotidesequences capable of interacting with transcription factors.

Promoter variant: The term “promoter variant” means a promotercomprising an alteration, i.e., a substitution, insertion, and/ordeletion, at one or more (e.g., several) positions. A substitution meansreplacement of the nucleotide occupying a position with a differentnucleotide; a deletion means removal of the nucleotide occupying aposition; and an insertion means adding a nucleotide adjacent to andimmediately following the nucleotide occupying a position. The term“promoter variant” will also encompass natural variants and in vitrogenerated variants obtained using methods well known in the art such asclassical mutagenesis, site-directed mutagenesis, and DNA shuffling.

Sequence identity: The relatedness between two amino acid sequences orbetween two polynucleotide sequences is described by the parameter“sequence identity”.

For purposes of the present invention, the sequence identity between twoamino acid sequences is determined using the Needleman-Wunsch algorithm(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implementedin the Needle program of the EMBOSS package (EMBOSS: The EuropeanMolecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), preferably version 3.0.0, 5.0.0, or later. The parametersused are gap open penalty of 10, gap extension penalty of 0.5, and theEBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The outputof Needle labeled “longest identity” (obtained using the—nobrief option)is used as the percent identity and is calculated as follows:(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the sequence identity between twodeoxyribonucleotide sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, supra) as implemented in theNeedle program of the EMBOSS package (EMBOSS: The European MolecularBiology Open Software Suite, Rice et al., 2000, supra), preferablyversion 3.0.0, 5.0.0, or later. The parameters used are gap open penaltyof 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version ofNCBI NUC4.4) substitution matrix. The output of Needle labeled “longestidentity” (obtained using the—nobrief option) is used as the percentidentity and is calculated as follows:(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Subsequence: The term “subsequence” means a polynucleotide having one ormore (e.g., several) nucleotides absent from the 5′ and/or 3′ end of amature polypeptide coding sequence wherein the subsequence encodes afragment having biological activity, or absent from the 5′ and/or 3′ endof a promoter sequence wherein the promoter subsequence has promoteractivity. In one aspect, the subsequence has at least 85%, e.g., atleast 90% or at least 95% of the number of nucleotides as the maturepolypeptide coding sequence. In another aspect, the promoter subsequencehas at least 85%, e.g., at least 90% or at least 95% of the number ofnucleotides as the promoter sequence.

Tandem promoter: The term “tandem promoter” means two or more (e.g.,several) promoters linked in tandem, each of which is operably linked toa coding sequence and mediates the transcription of the coding sequenceinto mRNA.

Very high stringency conditions: The term “very high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 70° C.

Very low stringency conditions: The term “very low stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 25% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 45° C.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods for producing a polypeptide,comprising: (a) cultivating a fungal host cell in a medium conducive forthe production of the polypeptide, wherein the fungal host cellcomprises a polynucleotide encoding the polypeptide operably linked to apromoter selected from the group consisting of (i) a promoter comprisinga nucleotide sequence having at least 60% sequence identity to SEQ IDNO: 1; (ii) a promoter comprising a nucleotide sequence that hybridizesunder at least medium stringency conditions with SEQ ID NO: 1 or thefull-length complement thereof; (iii) a promoter comprising SEQ ID NO:1; (iv) a promoter comprising a subsequence of (i), (ii), or (iii) thatretains promoter activity; and (v) a mutant, hybrid, or tandem promoterof (i), (ii), (iii), or (iv); wherein the polynucleotide encoding thepolypeptide is foreign to the promoter; and (b) isolating thepolypeptide from the cultivation medium.

In the production methods of the present invention, the cells arecultivated in a nutrient medium suitable for production of thepolypeptide using methods known in the art. For example, the cell may becultivated by shake flask cultivation, or small-scale or large-scalefermentation (including continuous, batch, fed-batch, or solid statefermentations) in laboratory or industrial fermentors in a suitablemedium and under conditions allowing the polypeptide to be expressedand/or isolated. The cultivation takes place in a suitable nutrientmedium comprising carbon and nitrogen sources and inorganic salts, usingprocedures known in the art. Suitable media are available fromcommercial suppliers or may be prepared according to publishedcompositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

The polypeptide may be detected using methods known in the art that arespecific for the polypeptide. These detection methods may include use ofspecific antibodies, high performance liquid chromatography, capillarychromatography, formation of an enzyme product, disappearance of anenzyme substrate, or SDS-PAGE. For example, an enzyme assay may be usedto determine the activity of an enzyme. Procedures for determiningenzyme activity are known in the art for many enzymes (see, for example,D. Schomburg and M. Salzmann (eds.), Enzyme Handbook, Springer-Verlag,New York, 1990).

The polypeptide may be recovered using methods known in the art. Forexample, the polypeptide may be recovered from the nutrient medium byconventional procedures including, but not limited to, collection,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation. In one aspect, the whole fermentation broth is recovered.

The isolated polypeptide may then be further purified by a variety ofprocedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing), differential solubility (e.g.,ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g.,Protein Purification, Janson and Ryden, editors, VCH Publishers, NewYork, 1989) to obtain substantially pure polypeptides.

Promoters

The present invention also relates to isolated promoters selected fromthe group consisting of (i) a promoter comprising a nucleotide sequencehaving at least 60% sequence identity to SEQ ID NO: 1; (ii) a promotercomprising a nucleotide sequence that hybridizes under at least mediumstringency conditions with SEQ ID NO: 1 or the full-length complementthereof; (iii) a promoter comprising SEQ ID NO: 1; (iv) a promotercomprising a subsequence of (i), (ii), or (iii) that retains promoteractivity; and (v) a mutant, hybrid, or tandem promoter of (i), (ii),(iii), or (iv); and to constructs, vectors, and fungal host cellscomprising the promoter operably linked to a polynucleotide encoding apolypeptide.

In one aspect, the isolated promoters have a sequence identity to SEQ IDNO: 1 of at least 60%, e.g., at least 65%, at least 70%, at least 75%,at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100%, which have promoter activity.

In one embodiment, a promoter of the present invention comprises orconsists of the nucleotide sequence of SEQ ID NO: 1 or an allelicvariant thereof; or is a subsequence thereof having promoter activity.In another aspect, the promoter comprises or consists of the nucleotidesequence of SEQ ID NO: 1.

A subsequence of SEQ ID NO: 1 (i.e., a truncated promoter) comprises atruncation at the 5′ end so that the sequence at the 3′ end closest tothe ATG codon is maintained. The subsequence can be at least 600nucleotides, e.g., at least 700 nucleotides, at least 750 nucleotides,at least 800 nucleotides, at least 850 nucleotides, or at least 900nucleotides, that has promoter activity.

In one aspect, the isolated promoters comprise nucleotide sequences thathybridize under very low stringency conditions, low stringencyconditions, medium stringency conditions, medium-high stringencyconditions, high stringency conditions, or very high stringencyconditions with SEQ ID NO: 1 or a subsequence thereof; or thefull-length complement of the foregoing (J. Sambrook, E. F. Fritsch, andT. Maniatis, 1989, Molecular Cloning, A Laboratory Manual, 2d edition,Cold Spring Harbor, N.Y.).

The polynucleotide of SEQ ID NO: 1, or a subsequence thereof, may beused to design nucleic acid probes to identify and clone promoter DNAfrom strains of different genera or species according to methods wellknown in the art. In particular, such probes can be used forhybridization with the genomic DNA or cDNA of the genus or species ofinterest, following standard Southern blotting procedures, in order toidentify and isolate the corresponding promoter DNA therein. Such probescan be considerably shorter than the entire sequence, but should be atleast 15, e.g., at least 25, at least 35, or at least 70 nucleotides inlength. Preferably, the nucleic acid probe is at least 100 nucleotidesin length, e.g., at least 200 nucleotides, at least 300 nucleotides, atleast 400 nucleotides, at least 500 nucleotides, at least 600nucleotides, at least 700 nucleotides, at least 800 nucleotides, or atleast 900 nucleotides in length. Both DNA and RNA probes can be used.The probes are typically labeled for detecting the correspondingpromoter DNA (for example, with ³²P, ³H, ³⁵S, biotin, or avidin). Suchprobes are encompassed by the present invention.

A genomic DNA or cDNA library prepared from such other strains may bescreened for DNA that hybridizes with the probes described herein andhas promoter activity. Genomic or other DNA from such other strains maybe separated by agarose or polyacrylamide gel electrophoresis, or otherseparation techniques. DNA from the libraries or the separated DNA maybe transferred to and immobilized on nitrocellulose or other suitablecarrier material. In order to identify a clone or DNA that is homologouswith SEQ ID NO: 1, or a subsequence thereof, the carrier material ispreferably used in a Southern blot.

For purposes of the present invention, hybridization indicates that thepolynucleotide hybridizes to a labeled nucleic acid probe correspondingto SEQ ID NO: 1 or the full-length complement thereof, or a subsequencethereof, under very low to very high stringency conditions. Molecules towhich the nucleic acid probe hybridizes under these conditions can bedetected using, for example, X-ray film.

In one embodiment, the nucleic acid probe is SEQ ID NO: 1 or asubsequence thereof. In another embodiment, the nucleic acid probe isSEQ ID NO: 1.

For short probes of about 15 nucleotides to about 70 nucleotides inlength, stringency conditions are defined as prehybridization andhybridization at about 5° C. to about 10° C. below the calculated T_(m)using the calculation according to Bolton and McCarthy (1962, Proc.Natl. Acad. Sci. USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6mM EDTA, 0.5% NP-40, 1×Denhardt's solution, 1 mM sodium pyrophosphate, 1mM sodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA perml following standard Southern blotting procedures for 12 to 24 hoursoptimally. The carrier material is finally washed once in 6×SCC plus0.1% SDS for 15 minutes and twice each for 15 minutes using 6×SSC at 5°C. to 10° C. below the calculated T_(m).

In another aspect, the isolated promoters may be mutants of a promotercomprising the polynucleotide sequence of SEQ ID NO: 1, or a subsequencethereof, that has promoter activity. The mutant promoters comprise oneor more mutations (e.g., several) of SEQ ID NO: 1, or a subsequencethereof, that has promoter activity. Each mutation is an independentsubstitution, deletion, and/or insertion of a nucleotide. Theintroduction of a substitution, deletion, and/or insertion of anucleotide into the promoter may be accomplished using any of themethods known in the art such as classical mutagenesis, site-directedmutagenesis, or DNA shuffling. Particularly useful is a procedure whichutilizes a supercoiled, double stranded DNA vector with an insert ofinterest and two synthetic primers containing the desired mutation. Theoligonucleotide primers, each complementary to opposite strands of thevector, extend during temperature cycling by means of Pfu DNApolymerase. On incorporation of the primers, a mutated plasmidcontaining staggered nicks is generated. Following temperature cycling,the product is treated with Dpn I which is specific for methylated andhemimethylated DNA to digest the parental DNA template and to select formutation-containing synthesized DNA. Other procedures known in the artmay also be used.

In another aspect, the isolated promoters may be hybrid promoterscomprising a portion of a promoter of the present invention and aportion of another promoter, e.g., a leader sequence of one promoter andthe transcription start site from the other promoter; or a portion ofone or more (e.g., several) promoters of the present invention and aportion of one or more (e.g., several) other promoters. The otherpromoter may be any promoter sequence which shows transcriptionalactivity in the fungal host cell of choice including a mutant,truncated, and hybrid promoter, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell. The other promoter sequence may also be aportion of a promoter of the present invention. The other promotersequence may also be native or foreign to the polynucleotide encodingthe polypeptide and native or foreign to the cell.

In another aspect, the isolated promoters may be tandem promoterscomprising one or more (e.g., several) promoters of the presentinvention and one or more (e.g., several) other promoters. The one ormore (e.g., several) other promoters may be promoters of the presentinvention. The one or more (e.g., several) other promoters may bepromoters such as those exemplified below. Two or more (e.g., several)promoter sequences of the tandem promoter may simultaneously promote thetranscription of the polynucleotide. Alternatively, one or more (e.g.,several) of the promoter sequences of the tandem promoter may promotethe transcription of the polynucleotide at different stages of growth ofthe cell. In one embodiment, the tandem promoter comprises twopromoters. In another embodiment, the tandem promoter comprises threepromoters. In another embodiment, the tandem promoter comprises fourpromoters. In another embodiment, the tandem promoter comprises fivepromoters.

Examples of other promoters useful in the construction of tandempromoters or hybrid promoters with the promoters of the presentinvention include the promoters obtained from the genes for Aspergillusnidulans acetamidase, Aspergillus niger neutral alpha-amylase,Aspergillus niger acid stable alpha-amylase, Aspergillus niger orAspergillus awamori glucoamylase (glaA), Aspergillus oryzae TAKAamylase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triosephosphate isomerase, Fusarium oxysporum trypsin-like protease (WO96/00787), Fusarium venenatum amyloglucosidase (WO 00/56900), Fusariumvenenatum Daria (WO 00/56900), Fusarium venenatum Quinn (WO 00/56900),Rhizomucor miehei lipase, Rhizomucor miehei aspartic proteinase,Trichoderma reesei beta-glucosidase, Trichoderma reeseicellobiohydrolase I, Trichoderma reesei cellobiohydrolase II,Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II,Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanaseV, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II,Trichoderma reesei xylanase III, Trichoderma reesei beta-xylosidase, andTrichoderma reesei translation elongation factor, NA2-tpi promoter (amodified promoter from an Aspergillus neutral alpha-amylase gene inwhich the untranslated leader has been replaced by an untranslatedleader from an Aspergillus triose phosphate isomerase gene; non-limitingexamples include modified promoters from an Aspergillus niger neutralalpha-amylase gene in which the untranslated leader has been replaced byan untranslated leader from an Aspergillus nidulans or Aspergillusoryzae triose phosphate isomerase gene); Saccharomyces cerevisiaeenolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1),Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase; and mutant, truncated, and hybrid promotersthereof. Other promoters are described in U.S. Pat. No. 6,011,147 and byRomanos et al., 1992, Yeast 8: 423-488.

In the methods of the present invention, a hybrid or tandem promoter ofthe present invention will be understood to be foreign to apolynucleotide encoding a polypeptide even if the wild-type promoter isnative to the polynucleotide. For example, in a tandem promoterconsisting of at least two promoters, one of the promoters may be thewild-type promoter of the polynucleotide encoding the polypeptide.

Polypeptides

The term “polypeptide” is defined herein as a polypeptide encoded by acoding sequence that is foreign to a promoter of the present invention.

The polypeptide may be any polypeptide having a biological activity ofinterest. The term “polypeptide” is not meant herein to refer to aspecific length of the encoded product and, therefore, encompassespeptides, oligopeptides, and proteins. The term “polypeptide” alsoencompasses polypeptides, which comprise a combination of partial and/orcomplete polypeptide sequences obtained from at least two differentpolypeptides wherein one or more (e.g., several) may be heterologous tothe fungal cell. Polypeptides further include naturally occurringallelic and engineered variations of a polypeptide.

In one aspect, the polypeptide is an antibody, antigen, antimicrobialpeptide, enzyme, growth factor, hormone, immunodilator,neurotransmitter, receptor, reporter protein, structural protein, andtranscription factor.

In one embodiment, the enzyme is an oxidoreductase, transferase,hydrolase, lyase, isomerase, or ligase. In another embodiment, theenzyme is an acetylmannan esterase, acetylxylan esterase,alpha-galactosidase, alpha-glucosidase, alpha-glucuronidase,aminopeptidase, amylase, amyloglucosidase, arabinanase,arabinofuranosidase, beta-galactosidase, beta-glucosidase,beta-xylosidase, carbohydrase, carboxypeptidase, catalase,cellobiohydrolase, cellulase, chitinase, coumaric acid esterase,cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease,endoglucanase, esterase, feruloyl esterase, GH61 polypeptide havingcellulolytic enhancing activity, glucocerebrosidase, glucuronidase,hemicellulase, invertase, laccase, lipase, mannanase, mannosidase,mutanase, oxidase, pectinolytic enzyme, peroxidase, phospholipase,phytase, polyphenoloxidase, protease, ribonuclease, transglutaminase,urokinase, or xylanase.

In another aspect, the polypeptide is an albumin, collagen,tropoelastin, elastin, or gelatin.

In another aspect, the polypeptide is an expansin or a swollenin.

In another aspect, the polypeptide is hybrid polypeptide in which aregion of one polypeptide is fused at the N-terminus or the C-terminusof a region of another polypeptide.

In another aspect, the polypeptide is a chimeric polypeptide in whichone or more (e.g., several) regions of one polypeptide are replaced withone or more (e.g., several) regions from one or more (e.g., several)other polypeptides.

In another aspect, the polypeptide is a fusion polypeptide or cleavablefusion polypeptide in which one polypeptide is fused at the N-terminusor the C-terminus of another polypeptide. A fusion polypeptide isproduced by fusing a polynucleotide encoding the one polypeptide to apolynucleotide encoding the other polypeptide. Techniques for producingfusion polypeptides are known in the art, and include ligating thecoding sequences encoding the polypeptides so that they are in frame andthat expression of the fusion polypeptide is under control of the samepromoter(s) and terminator. Fusion polypeptides may also be constructedusing intein technology in which fusion polypeptides are createdpost-translationally (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawsonet al., 1994, Science 266: 776-779).

A fusion polypeptide can further comprise a cleavage site between thetwo polypeptides. Upon secretion of the fusion protein, the site iscleaved releasing the two polypeptides. Examples of cleavage sitesinclude, but are not limited to, the sites disclosed in Martin et al.,2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000,J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl.Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton etal., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995,Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure,Function, and Genetics 6: 240-248; and Stevens, 2003, Drug DiscoveryWorld 4: 35-48.

The polynucleotide encoding a polypeptide may be obtained from anyprokaryotic, eukaryotic, or other source. For purposes of the presentinvention, the term “obtained from” as used herein in connection with agiven source shall mean that the polypeptide is produced by the sourceor by a cell in which a gene from the source has been inserted.

The techniques used to isolate or clone a polynucleotide encoding apolypeptide are known in the art and include isolation from genomic DNA,preparation from cDNA, or a combination thereof. The cloning of thepolynucleotide from such genomic DNA can be effected, e.g., by using thepolymerase chain reaction (PCR). See, for example, Innis et al., 1990,PCR Protocols: A Guide to Methods and Application, Academic Press, NewYork. The cloning procedures may involve excision and isolation of adesired nucleic acid fragment comprising the polynucleotide encoding thepolypeptide, insertion of the fragment into a vector molecule, andincorporation of the recombinant vector into the fungal cell wheremultiple copies or clones of the polynucleotide will be replicated. Thepolynucleotide may be of genomic, cDNA, RNA, semisynthetic, syntheticorigin, or any combinations thereof.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide encoding a polypeptide operably linked to a promoter ofthe present invention and one or more (e.g., several) control sequenceswhich direct the expression of the coding sequence in a suitable hostcell under conditions compatible with the control sequences. Expressionwill be understood to include any step involved in the production of thepolypeptide including, but not limited to, transcription,post-transcriptional modification, translation, post-translationalmodification, and secretion.

A polynucleotide may be manipulated in a variety of ways to provide forexpression of the polypeptide. Manipulation of the polynucleotide priorto its insertion into a vector may be desirable or necessary dependingon the expression vector. The techniques for modifying a polynucleotideutilizing recombinant DNA methods are well known in the art.

The control sequence may also be a transcription terminator, which isrecognized by a host cell to terminate transcription. The terminator isoperably linked to the 3′-terminus of the polynucleotide encoding thepolypeptide. Any terminator that is functional in the host cell may beused in the present invention.

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus nidulans acetamidase, Aspergillusnidulans anthranilate synthase, Aspergillus niger glucoamylase,Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase,Fusarium oxysporum trypsin-like protease, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I,Trichoderma reesei xylanase II, Trichoderma reesei xylanase III,Trichoderma reesei beta-xylosidase, and Trichoderma reesei translationelongation factor.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be a leader, a nontranslated region of anmRNA that is important for translation by the host cell. The leader isoperably linked to the 5′-terminus of the polynucleotide encoding thepolypeptide. Any leader that is functional in the host cell may be used.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the polynucleotide and, whentranscribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell may be used.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus nidulans anthranilatesynthase, Aspergillus niger glucoamylase, Aspergillus nigeralpha-glucosidase Aspergillus oryzae TAKA amylase, and Fusariumoxysporum trypsin-like protease.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a polypeptide anddirects the polypeptide into the cell's secretory pathway. The 5′-end ofthe coding sequence of the polynucleotide may inherently contain asignal peptide coding sequence naturally linked in translation readingframe with the segment of the coding sequence that encodes thepolypeptide. Alternatively, the 5′-end of the coding sequence maycontain a signal peptide coding sequence that is foreign to the codingsequence. A foreign signal peptide coding sequence may be required wherethe coding sequence does not naturally contain a signal peptide codingsequence. Alternatively, a foreign signal peptide coding sequence maysimply replace the natural signal peptide coding sequence in order toenhance secretion of the polypeptide. However, any signal peptide codingsequence that directs the expressed polypeptide into the secretorypathway of a host cell may be used.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus niger neutral amylase, Aspergillus niger glucoamylase,Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicolainsolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucormiehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a polypeptide. Theresultant polypeptide is known as a proenzyme or propolypeptide (or azymogen in some cases). A propolypeptide is generally inactive and canbe converted to an active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Myceliophthorathermophila laccase (WO 95/33836), Rhizomucor miehei asparticproteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present, thepropeptide sequence is positioned next to the N-terminus of apolypeptide and the signal peptide sequence is positioned next to theN-terminus of the propeptide sequence.

It may also be desirable to add regulatory sequences that regulateexpression of the polypeptide relative to the growth of the host cell.Examples of regulatory sequences are those that cause expression of thegene to be turned on or off in response to a chemical or physicalstimulus, including the presence of a regulatory compound. In yeast, theADH2 system or GAL1 system may be used. In filamentous fungi, theAspergillus niger glucoamylase promoter, Aspergillus oryzae TAKAalpha-amylase promoter, and Aspergillus oryzae glucoamylase promoter,Trichoderma reesei cellobiohydrolase I promoter, and Trichoderma reeseicellobiohydrolase II promoter may be used. Other examples of regulatorysequences are those that allow for gene amplification. In eukaryoticsystems, these regulatory sequences include the dihydrofolate reductasegene that is amplified in the presence of methotrexate, and themetallothionein genes that are amplified with heavy metals. In thesecases, the polynucleotide encoding the polypeptide would be operablylinked to the regulatory sequence.

The present invention also relates to nucleic acid constructs foraltering the expression of a gene encoding a polypeptide which isendogenous to a host cell. The constructs may contain the minimal numberof components necessary for altering expression of the endogenous gene.In one embodiment, the nucleic acid constructs preferably comprise (a) atargeting sequence, (b) a promoter of the present invention, (c) anexon, and (d) a splice-donor site. Upon introduction of the nucleic acidconstruct into a cell, the construct inserts by homologous recombinationinto the cellular genome at the endogenous gene site. The targetingsequence directs the integration of elements (a)-(d) into the endogenousgene such that elements (b)-(d) are operably linked to the endogenousgene. In another embodiment, the nucleic acid constructs comprise (a) atargeting sequence, (b) a promoter of the present invention, (c) anexon, (d) a splice-donor site, (e) an intron, and (f) a splice-acceptorsite, wherein the targeting sequence directs the integration of elements(a)-(f) such that elements (b)-(f) are operably linked to the endogenousgene. However, the constructs may contain additional components such asa selectable marker.

In both embodiments, the introduction of these components results inproduction of a new transcription unit in which expression of theendogenous gene is altered. In essence, the new transcription unit is afusion product of the sequences introduced by the targeting constructsand the endogenous gene. In one embodiment in which the endogenous geneis altered, the gene is activated. In this embodiment, homologousrecombination is used to replace, disrupt, or disable the regulatoryregion normally associated with the endogenous gene of a parent cellthrough the insertion of a regulatory sequence which causes the gene tobe expressed at higher levels than evident in the corresponding parentcell. The activated gene can be further amplified by the inclusion of anamplifiable selectable marker gene in the construct using methods wellknown in the art (see, for example, U.S. Pat. No. 5,641,670). In anotherembodiment in which the endogenous gene is altered, expression of thegene is reduced.

The targeting sequence can be within the endogenous gene, immediatelyadjacent to the gene, within an upstream gene, or upstream of and at adistance from the endogenous gene. One or more (e.g., several) targetingsequences can be used. For example, a circular plasmid or DNA fragmentpreferably employs a single targeting sequence, while a linear plasmidor DNA fragment preferably employs two targeting sequences.

The constructs further contain one or more (e.g., several) exons of theendogenous gene. An exon is defined as a DNA sequence which is copiedinto RNA and is present in a mature mRNA molecule such that the exonsequence is in-frame with the coding region of the endogenous gene. Theexons can, optionally, contain DNA which encodes one or more (e.g.,several) amino acids and/or partially encodes an amino acid.Alternatively, the exon contains DNA which corresponds to a 5′non-encoding region. Where the exogenous exon or exons encode one ormore (e.g., several) amino acids and/or a portion of an amino acid, thenucleic acid construct is designed such that, upon transcription andsplicing, the reading frame is in-frame with the coding region of theendogenous gene so that the appropriate reading frame of the portion ofthe mRNA derived from the second exon is unchanged.

The splice-donor site of the constructs directs the splicing of one exonto another exon. Typically, the first exon lies 5′ of the second exon,and the splice-donor site overlapping and flanking the first exon on its3′ side recognizes a splice-acceptor site flanking the second exon onthe 5′ side of the second exon. A splice-acceptor site, like asplice-donor site, is a sequence which directs the splicing of one exonto another exon. Acting in conjunction with a splice-donor site, thesplicing apparatus uses a splice-acceptor site to effect the removal ofan intron.

The present invention further relates to methods for producing apolypeptide comprising (a) cultivating a homologously recombinant cell,having incorporated therein a transcription unit comprising a promoterof the present invention, an exon, and/or a splice donor site operablylinked to a second exon of an endogenous polynucleotide encoding thepolypeptide, under conditions conducive for production of thepolypeptide, wherein the polynucleotide encoding the polypeptide isforeign to the promoter; and (b) recovering the polypeptide. The methodsare based on the use of gene activation technology, for example, asdescribed in U.S. Pat. No. 5,641,670.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a promoter of the present invention, a polynucleotideencoding a polypeptide, and transcriptional and translational stopsignals. The various nucleotide and control sequences may be joinedtogether to produce a recombinant expression vector that may include oneor more (e.g., several) convenient restriction sites to allow forinsertion or substitution of the polynucleotide encoding the polypeptideat such sites. Alternatively, the polynucleotide may be expressed byinserting the polynucleotide operably linked to a promoter of thepresent invention or a nucleic acid construct thereof into anappropriate vector for expression. In creating the expression vector,the coding sequence is located in the vector so that the coding sequenceis operably linked to a promoter of the present invention.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more selectable markers thatpermit easy selection of transformed, transfected, transduced, or thelike cells. A selectable marker is a gene the product of which providesfor biocide or viral resistance, resistance to heavy metals, prototrophyto auxotrophs, and the like.

Examples of selectable markers for yeast host cells include, but are notlimited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectablemarkers for use in a filamentous fungal host cell include, but are notlimited to, adeA (phosphoribosylaminoim idazole-succinocarboxam idesynthase), adeB (phosphoribosyl-aminoimidazole synthase), amdS(acetamidase), argB (ornithine carbamoyltransferase), bar(phosphinothricin acetyltransferase), hph (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof.Preferred for use in an Aspergillus cell are Aspergillus nidulans orAspergillus oryzae amdS and pyrG genes and a Streptomyces hygroscopicusbar gene. Preferred for use in a Trichoderma cell are adeA, adeB, amdS,hph, and pyrG genes.

The selectable marker may be a dual selectable marker system asdescribed in WO 2010/039889. In one aspect, the dual selectable markeris an hph-tk dual selectable marker system.

The vector preferably contains an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector may containadditional polynucleotides for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should contain a sufficientnumber of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000base pairs, and 800 to 10,000 base pairs, which have a high degree ofsequence identity to the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding polynucleotides. On the other hand, the vectormay be integrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANS1 (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of theAMA1 gene and construction of plasmids or vectors comprising the genecan be accomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide may be inserted into a host cellto increase production of a polypeptide. An increase in the copy numberof the polynucleotide can be obtained by integrating at least oneadditional copy of the sequence into the host cell genome or byincluding an amplifiable selectable marker gene with the polynucleotidewhere cells containing amplified copies of the selectable marker gene,and thereby additional copies of the polynucleotide, can be selected forby cultivating the cells in the presence of the appropriate selectableagent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga promoter of the present invention operably linked to a polynucleotideencoding a polypeptide, which are advantageously used in the recombinantproduction of the polypeptide. A vector comprising a promoter of thepresent invention operably linked to a polynucleotide encoding apolypeptide is introduced into a host cell so that the vector ismaintained as a chromosomal integrant or as a self-replicatingextra-chromosomal vector as described earlier. The term “host cell”encompasses any progeny of a parent cell that is not identical to theparent cell due to mutations that occur during replication. The choiceof a host cell will to a large extent depend upon the gene encoding thepolypeptide and its source.

The host cell may be any fungal cell useful in the methods of thepresent invention. Fungi” as used herein includes the phyla Ascomycota,Basidiomycota, Chytridiomycota, and Zygomycota as well as the Oomycotaand all mitosporic fungi (as defined by Hawksworth et al., In, Ainsworthand Bisby's Dictionary of The Fungi, 8th edition, 1995, CABInternational, University Press, Cambridge, UK).

The fungal host cell may be a yeast cell. “Yeast” as used hereinincludes ascosporogenous yeast (Endomycetales), basidiosporogenousyeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes).Since the classification of yeast may change in the future, for thepurposes of this invention, yeast shall be defined as described inBiology and Activities of Yeast (Skinner, Passmore, and Davenport,editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cell such as aKluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomycesoviformis, or Yarrowia lipolytica cell.

The fungal host cell may be a filamentous fungal cell. “Filamentousfungi” include all filamentous forms of the subdivision Eumycota andOomycota (as defined by Hawksworth et al., 1995, supra). The filamentousfungi are generally characterized by a mycelial wall composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiae is by budding of a unicellular thallus andcarbon catabolism may be fermentative.

The filamentous fungal host cell may be an Acremonium, Aspergillus,Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus,Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Phiebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergillusawamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea,Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora,Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium merdarium, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporiumzonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsuiphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell.

In one aspect, the host cell is a Trichoderma host cell (e.g., aTrichoderma reesei host cell).

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422.Suitable methods for transforming Fusarium species are described byMalardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may betransformed using the procedures described by Becker and Guarente, InAbelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology, Volume 194, pp 182-187,Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

The present invention is further described by the following exampleswhich should not be construed as limiting the scope of the invention.

EXAMPLES

Strains

Trichoderma reesei RutC30 (ATCC 56765; Montenecourt and Eveleigh, 1979,Adv. Chem. Ser. 181: 289-301) was derived from Trichoderma reesei Qm6A(ATCC 13631; Mandels and Reese, 1957, J. Bacteriol. 73: 269-278).

Trichoderma reesei RutC30-2 is a mutagenized strain of Trichodermareesei RutC30 (ATCC 56765; Montenecourt and Eveleigh, 1979, supra).

Media and Solutions

YP medium was composed of 10 g of yeast extract, 20 g of bacto tryptone,and deionized water to 1 liter.

Cellulase-inducing medium was composed of 20 g of cellulose, 10 g ofcorn steep solids, 1.45 g of (NH₄)₂SO₄, 2.08 g of KH₂PO₄, 0.28 g ofCaCl₂, 0.42 g of MgSO₄.7H₂O, 0.42 ml of trace metals solution, anddeionized water to 1 liter.

Trace metals solution was composed of 216 g of FeCl₃.6H₂O, 58 g ofZnSO₄.7H₂O, 27 g of MnSO₄.H₂O, 10 g of CuSO₄.5H₂O, 2.4 g of H₃BO₃, 336 gof citric acid, and deionized water to 1 liter.

STC buffer was composed of 1 M sorbitol, 10 mM CaCl₂, and 10 mMTris-HCl, pH 7.5.

COVE plates were composed of 342 g of sucrose, 10 ml of COVE saltssolution, 10 ml of 1 M acetamide, 10 ml of 1.5 M CsCl, 25 g of Nobleagar, and deionized water to 1 liter.

COVE salts solution was composed of 26 g of KCl, 26 g of MgSO₄, 76 g ofKH₂PO₄, 50 ml of COVE trace metals solution, and deionized water to 1liter.

COVE trace metals solution was composed of 0.04 g of Na₂B₄O₇.10H₂O, 0.4g of CuSO₄.5H₂O, 1.2 g of FeSO₄.7H₂O, 0.7 g of MnSO₄.H₂O, 0.8 g ofNa₂MoO₂.H₂O, 10 g of ZnSO₄.7H₂O, and deionized water to 1 liter.

COVE2 plates were composed of 30 g of sucrose, 20 ml of COVE saltssolution, 25 g of Noble agar, 10 ml of 1 M acetamide, and deionizedwater to 1 liter.

PDA plates were composed of 39 grams of potato dextrose agar anddeionized water to 1 liter.

2XYT-Amp plates were composed of 10 g of tryptone, 5 g of yeast extract,5 g of sodium chloride, 15 g of bacto agar, 100 mg and ampicillin, anddeionized water to 1 liter.

LB medium was composed of 10 g of tryptone, 5 g of yeast extract, 5 g ofsodium chloride, and deionized water to 1 liter.

SOC medium was composed of 2% tryptone, 0.5% yeast extract, 10 mM NaCl,2.5 mM KCl, 10 mM MgCl₂, 10 mM MgSO₄, and 20 mM glucose in deionizedwater.

TAE buffer was composed of 4.84 g of Tris base, 1.14 ml of glacialacetic acid, 2 ml of 0.5 M EDTA pH 8.0, and deionized water to 1 liter.

TE buffer was composed of 10 mM Tris Base and 1 mM EDTA, pH 8.0.

Example 1 Construction of Reporter Plasmid pSMai226

Reporter plasmid pSMai226 was constructed, as described below. toanalyze the strength of Trichoderma reesei promoters using anAspergillus fumigatus beta-glucosidase as a reporter.

The Aspergillus fumigatus beta-glucosidase coding region and theTrichoderma reesei cellobiohydrolase I (cbh1/cel7a) terminator wereamplified from pEJG107 (WO 2005/047499) using primers 069448 and 069449shown below. The underlined text represents an introduced Sph I site andthe italicized text represents a region with homology to the site ofinsertion in plasmid pMJ09 (WO 2005/056772) for IN-FUSION™ cloning(Clontech Laboratories Inc., Mountain View, Calif., USA).

069448: (SEQ ID NO: 2) 5′-GAATTGTTTAAACGTCGAC GCATGCATGAGATTCGGTTGGCTCGAGGTGGC-3′ 069449: (SEQ ID NO: 3)5′-CGAAATGGATTGATTGTCTACCGCCAGGTGTCAGT-3′

The amplification reaction (50 μl) was composed of 25 ng of pEJG107, 200μM dNTP each, 0.4 μM primer 069448, 0.4 μM primer 069449, 1× PHUSION®Buffer (New England Biolabs, Ipswich, Mass., USA) and 1 unit of PHUSION®Hot Start High Fidelity DNA polymerase (New England Biolabs, Ipswich,Mass., USA). The reaction was incubated in an EPPENDORF® MASTERCYCLER®(Eppendorf A G, Hamburg, Germany) programmed for 1 cycle at 98° C. for30 seconds; 30 cycles each at 98° C. for 30 seconds, 60° C. for 30seconds, and 72° C. for 3 minutes 30 seconds; and 1 cycle at 72° C. for15 minutes.

The reaction products were separated by 1% agarose gel electrophoresisusing TAE buffer where a 3610 bp fragment was excised from the gel andpurified using a MINELUTE® Gel Extraction Kit (QIAGEN Inc., Valencia,Calif., USA) according to the manufacturer's instructions.

Plasmid pMJ09 was digested with Acc I where a 5.6 kb fragment comprisingthe Aspergillus nidulans fungal acetamidase gene (amdS) was purified by1% agarose gel electrophoresis using TAE buffer, excised from the gel,and purified using a MINELUTE® Gel Extraction Kit according to themanufacturer's instructions.

The 3610 bp PCR fragment was then ligated to the 5.6 kb fragment of AccI digested pMJ09 using an IN-FUSION™ ADVANTAGE™ PCR Cloning Kit(Clontech Laboratories Inc., Mountain View, Calif., USA) according tothe manufacturer's instructions to provide pSMai226 (FIG. 1). Theligation reaction mixture (10 μl) was composed of 1× IN-FUSION™ buffer,1 μl of IN-FUSION™ enzyme, 100 ng of digested pMJ09 fragment, and 63.5ng of the purified 3610 bp PCR fragment. The reaction mixture wasincubated at 37° C. for 15 minutes, followed by 15 minutes at 50° C.After diluting the reaction mixture with 50 μl of TE buffer (pH 8), 2.5μl of the reaction was used to transform E. coli ONE SHOT® TOP10competent cells according to the manufacturer's protocol (Invitrogen,Carlsbad, Calif., USA). An E. coli transformant containing pSMai226 wasdetected by restriction digestion with Sph I and Spe I and plasmid DNAwas prepared using a BIOROBOT® 9600 (QIAGEN Inc., Valencia, Calif.,USA). The Aspergillus fumigatus beta-glucosidase gene and theTrichoderma reesei cellobiohydrolase I terminator insert in pSMai226were confirmed by DNA sequencing.

Example 2 Trichoderma reesei Strain RutC30 Genomic DNA Extraction

Trichoderma reesei strain RutC30 was grown in 50 ml of YEG medium in abaffled shake flask at 28° C. for 2 days with agitation at 200 rpm.Mycelia were harvested by filtration using MIRACLOTH® (Calbiochem, LaJolla, Calif., USA), washed twice in deionized water, and frozen underliquid nitrogen. Frozen mycelia were ground, by mortar and pestle, to afine powder, and total DNA was isolated using a DNEASY® Plant Maxi Kit(QIAGEN Inc., Valencia, Calif., USA).

Example 3 Construction of Plasmid pSaMe-CatP

Plasmid pSaMe-CatP was constructed, as described below, to comprise theTrichoderma reesei catalase promoter, the Aspergillus fumigatusbeta-glucosidase coding sequence, and the Trichoderma reeseicellobiohydrolase I gene terminator.

The 872 bp Trichoderma reesei catalase promoter region was amplifiedfrom Trichoderma reesei RutC30 genomic DNA using primers 069728 and069729 shown below. The italicized text represents a region withhomology to the site of insertion in plasmid pSMai226 for IN-FUSION™cloning.

Primer 069728: (SEQ ID NO: 4)5′-AACGTCGACGCATGCTTGTGCACGGCTTTGGGTTAGCAAA-3′ Primer 069729:(SEQ ID NO: 5) 5′-CCAACCGAATCTCATTTTTGCTGTTCTCGTTGGTT-3′

The amplification reaction (50 μl) was composed of Pfx AmplificationBuffer (Invitrogen, Carlsbad, Calif., USA), 200 μM dNTP each, 50 ng ofT. reesei RutC30 genomic DNA, 0.4 μM primer 069728, 0.4 μM primer069729, 25 mM MgSO₄, and 2.5 units of Pfx DNA polymerase (Invitrogen,Carlsbad, Calif., USA). The reactions were incubated in an EPPENDORF®MASTERCYCLER® 5333 programmed for 1 cycle at 95° C. for 2 minutes; 30cycles each at 95° C. for 15 seconds, 55° C. for 30 seconds, and 68° C.for 1 minute; and one cycle at 68° C. for 7 minutes.

The PCR reaction products were separated by 1% agarose gelelectrophoresis using TAE buffer where the 872 bp catalase promoter wasexcised from the gel and purified using a MINELUTE® Gel Extraction Kitaccording to the manufacturer's instructions.

Plasmid pSMai226 was digested with Sph I where a 9.2 kb fragment waspurified by 1% agarose gel electrophoresis using TAE buffer, excisedfrom the gel, and purified using a MINELUTE® Gel Extraction Kitaccording to the manufacturer's instructions.

The 872 bp PCR fragment was then ligated to 9.2 kb fragment of the Sph Idigested pSMai226 fragment using an IN-FUSION™ ADVANTAGE™ PCR CloningKit according to the manufacturer's instructions to provide pSaMe-CatP(FIG. 2). The ligation reaction mixture (10 μl) was composed of 1×IN-FUSION™ Buffer, 1 μl of IN-FUSION™ enzyme, 200 ng of digestedpSMai226, and 36 ng of the purified 872 bp PCR product. The reactionmixture was incubated at 37° C. for 15 minutes, followed by 15 minutesat 50° C. After diluting the reaction mix with 50 μl of TE buffer (pH8), 2.5 μl of the reaction were used to transform E. coli SOLOPACK® Goldcompetent cells (Agilent Technologies, Palo Alto, Calif., USA) accordingmanufacturer's instructions. An E. coli transformant containingpSaMe-CatP was detected by digestion and plasmid DNA was prepared usinga BIOROBOT® 9600. The Trichoderma reesei catalase promoter insert inpSaMe-CatP was confirmed by DNA sequencing (SEQ ID NO: 1; see FIG. 3).

Example 4 Expression of the Aspergillus fumigatus Beta-GlucosidaseUtilizing the Trichoderma reesei Catalase Promoter

Plasmid pSaMe-CatP encoding the mature Aspergillus fumigatusbeta-glucosidase operably linked to the Trichoderma reesei catalasepromoter (Example 3) was introduced into Trichoderma reesei RutC30-2 byPEG-mediated transformation (Penttila et al., 1987, Gene 61: 155-164).The plasmid contained the Aspergillus nidulans amdS gene to enabletransformants to grow on acetamide as the sole nitrogen source.

Trichoderma reesei RutC30-2 was cultivated at 27° C. and 90 rpm in 25 mlof YP medium supplemented with 2% (w/v) glucose and 10 mM uridine for 17hours. Mycelia was collected by filtration using a Vacuum DrivenDisposable Filtration System (Millipore, Bedford, Mass., USA) and washedtwice with deionized water and twice with 1.2 M sorbitol. Protoplastswere generated by suspending the washed mycelia in 20 ml of 1.2 Msorbitol containing 15 mg/ml of GLUCANEX™ (Novozymes A/S, Bagsvrd,Denmark) and 0.36 units/ml of chitinase (Sigma Chemical Co., St. Louis,Mo., USA) and incubating for 15-25 minutes at 34° C. with gentle shakingat 90 rpm. The protoplasts were collected by centrifuging for 7 minutesat 400×g and washed twice with cold 1.2 M sorbitol. The protoplasts werecounted using a haemacytometer and re-suspended in STC to a finalconcentration of 1×10⁸ protoplasts per ml. Excess protoplasts werestored in a Cryo 1° C. Freezing Container (Nalgene, Rochester, N.Y.,USA) at −80° C.

Approximately 2 μg of pSaMe-CatP digested with Pme I was added to 100 μlof protoplast solution above and mixed gently, followed by addition of200 μl of PEG buffer, mixing, and incubation at 30° C. for 30 minutes.STC (3 ml) was then added with mixing, and the transformation solutionwas streaked onto COVE plates using Aspergillus nidulans amdS selection.The plates were incubated at 28° C. for 5-7 days. Transformants weresubcultured onto COVE2 plates and grown at 28° C. Twenty-onetransformants were selected and subcultured onto fresh plates containingacetamide and allowed to sporulate for 7 days at 28° C.

The selected Trichoderma reesei transformants were cultivated in 125 mlbaffled shake flasks containing 25 ml of cellulase-inducing medium at pH6.0 and incubated at 28° C. and 200 rpm for 5 days. Trichoderma reeseiRutC30-2 was run as a control. Culture broth samples were removed at day5 and 1 ml of each culture broth was centrifuged at 13,000×g for 5minutes and the supernatants transferred to new tubes and stored at 4°C. until assayed for beta-glucosidase activity.

Beta-glucosidase activity in the supernatants above was determined atambient temperature using 25 μl aliquots of culture supernatants,diluted 1:10 in 50 mM succinate pH 5.0, using 200 μl of 0.5 mg/mlp-nitrophenyl-beta-D-glucopyranoside as substrate in 50 mM succinate pH5.0. After 15 minutes of incubation the reaction was stopped by adding100 μl of 1 M Tris-HCl pH 8.0 and the absorbance was readspectrophotometrically at 405 nm. One unit of beta-glucosidase activitycorresponded to production of 1 μmole of p-nitrophenyl per minute perliter at pH 5.0 and ambient temperature. Aspergillus nigerbeta-glucosidase (Novozyme 188, Novozymes A/S, Bagsvrd, Denmark) wasused as an enzyme standard.

The results demonstrated that all assayed transformants showedbeta-glucosidase activities higher than the control or parental strain,Trichoderma reesei RutC30-2. Transformant SaMe-CatP-19 produced thehighest beta-glucosidase activity which was 10-fold higher than thecontrol strain.

SDS-PAGE was performed using CRITERION® Tris-HCl (8-16% resolving) gels(Bio-Rad Laboratories, Inc., Hercules, Calif., USA) with The CRITERION®System (Bio-Rad Laboratories, Inc., Hercules, Calif., USA). Fivemicroliters of each supernatant from day five were suspended in 2×concentration of Laemmli Sample Buffer (Bio-Rad Laboratories, Inc.,Hercules, Calif., USA) and boiled in the presence of 5%beta-mercaptoethanol for 3 minutes. The supernatant samples were loadedonto a polyacrylamide gel and subjected to electrophoresis with 1×Tris/Glycine/SDS as running buffer (Bio-Rad Laboratories, Inc.,Hercules, Calif., USA). The resulting gel was stained with BIO-SAFE®Coomassie Blue G250 protein stain (Bio-Rad Laboratories, Inc., Hercules,Calif., USA).

Twelve of the 21 Trichoderma reesei CatP transformants produced aprotein of approximately 110 kDa that was not visible in the Trichodermareesei RutC30-2 control. Transformant Trichoderma reesei SaMe-CatP-19produced the highest level of beta-glucosidase.

Example 5 Analysis of Top Transformants in Non-Cellulose ContainingMedium

In order to test the strength of the promoter under non-cellulaseinducing conditions the top three transformants were cultivated induplicate in 125 ml baffled shake flasks containing 25 ml of YP and 2%glucose medium and incubated at 28° C. and 200 rpm for 5 days.Trichoderma reesei RutC30-2 was run as a control. Culture broth sampleswere removed at day 5 and one ml sample of each culture broth wascentrifuged at 13,000×g for 5 minutes and each supernatant wastransferred to a new tube and stored at 4° C. until assayed forbeta-glucosidase activity.

Beta-glucosidase activity was determined as described above in Example4. The results demonstrated that all three transformants exhibitedhigher beta-glucosidase activity than the control strain Trichodermareesei RutC30-2.

SDS-PAGE was performed as described in Example 4. The resultsdemonstrated that the assay results correlated with the SDS-PAGE resultsby the presence of an approximately 110 kDa band.

Example 6 Single Spore Isolation of Transformant SaMe-CatP-19

Trichoderma reesei transformant SaMe-CatP-19 spores were plated onto aPDA plate and incubated for five days at 34° C. A small area of theconfluent spore plate was washed with 0.5 ml of 0.01% TWEEN® 80 toresuspend the spores. A 100 μl aliquot of the spore suspension wasdiluted to a final volume of 5 ml with 0.01% TWEEN® 80. The sporeconcentration was determined with a hemocytometer and diluted to a finalconcentration of 0.1 spores per microliter. A 200 μl aliquot of thespore dilution was plated onto 150 mm COVE2 plates and incubated for 2-3days at 28° C. Eight emerging colonies were excised from the plate andtransferred to PDA plates. After 3 days at 28° C. spores weresubcultured on new PDA plates and allowed to grow for an additional 5days at 28° C. Five of the confluent spore plates were used to inoculate125 ml baffled shake flasks containing 25 ml of cellulase-inducingmedium at pH 6.0 and incubated at 28° C. and 200 rpm for 5 days.Trichoderma reesei RutC30-2 was run as a control. Culture broth sampleswere removed at day 5 and 1 ml of each culture broth was centrifuged at13,000×g for 5 minutes in a micro-centrifuge and the supernatantstransferred to new tubes. Five microliters of each supernatant weremixed with an equal volume of 2× loading buffer (10%beta-mercaptoethanol) and loaded onto a 1.5 mm 8%-16% Tris-GlycineSDS-PAGE gel and stained with BIO-SAFE® Coomassie Blue G250 proteinstain. SDS-PAGE profiles of the culture broths showed four of the fivetransformants were capable of expressing the beta-glucosidase. Onetransformant, SaMe-CatP-19C, produced the highest yields.

Example 7 Fermentation of Transformant SaMe-CatP-19C

Trichoderma reesei SaMe-CatP-19C and control strain Trichoderma reeseiRutC30-2 were fermented in duplicate in two-liter fermentations andtested for beta-glucosidase activity. Spores of Trichoderma reeseiSaMe-CatP-19C and Trichoderma reesei RutC30-2 grown on PDA plates wereinoculated into 500 ml shake flasks containing 100 ml of shake flaskmedium composed of standard carbon and nitrogen sources. The flasks wereplaced into an orbital shaker at 28° C. for approximately 48 hours.Fifty ml of the culture was added to a three liter glass jacketedfermentor (Applikon Biotechnology, Inc., Foster City, Calif., USA)containing 1.8 liters of fermentation batch medium composed of standardcarbon and nitrogen sources. Fermentation feed medium composed ofstandard carbon and nitrogen sources was dosed at a rate of 0 to 4g/L/hr for a period of 185 hours. The fermentation vessel was maintainedat a temperature of 28° C. and pH was controlled using an Applikon 1030control system (Applikon Biotechnology, Inc., Foster City, Calif., USA)to a set-point of 4.5+/−0.1. Air was added to the vessel at a rate of 1vvm and the broth was agitated by Rushton impeller rotating at 1100 to1300 rpm. Following fermentation, whole broth was harvested from thevessel and centrifuged at 3000×g to remove the biomass. The supernatantwas sterile filtered and stored at 5 to 10° C.

The samples were assayed for beta-glucosidase activity as describedabove in Example 4. Trichoderma reesei SaMe-CatP-19C exhibited higherbeta-glucosidase activity than the control strain Trichoderma reeseiRutC30-2. Total protein levels of both Trichoderma reesei SaMe-CatP-19Cand the control strain Trichoderma reesei RutC30-2 were comparable.

The present invention is further described by the following numberedparagraphs:

[1] A method for producing a polypeptide, comprising: (a) cultivating afungal host cell in a medium conducive for the production of thepolypeptide, wherein the fungal host cell comprises a polynucleotideencoding the polypeptide operably linked to a promoter selected from thegroup consisting of (i) a promoter comprising a nucleotide sequencehaving at least 60% sequence identity to SEQ ID NO: 1, (ii) a promotercomprising a nucleotide sequence that hybridizes under at least mediumstringency conditions with SEQ ID NO: 1; or the full-length complementthereof; (iii) a promoter comprising SEQ ID NO: 1; (iv) a promotercomprising a subsequence of (i), (ii), or (iii) that retains promoteractivity; and (v) a mutant, hybrid, or tandem promoter of (i), (ii),(iii), or (iv); wherein the polynucleotide encoding the polypeptide isforeign to the promoter; and (b) isolating the polypeptide from thecultivation medium.

[2] The method of paragraph 1, wherein the promoter comprises anucleotide sequence having at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 81%, at least 82%, at least 83%, atleast 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% or 100% sequence identity to SEQ ID NO: 1.

[3] The method of paragraph 1, wherein the promoter comprises anucleotide sequence that hybridizes under medium stringency conditions,medium-high stringency conditions, high stringency conditions, or veryhigh stringency conditions with SEQ ID NO: 1 or the full-lengthcomplement thereof.

[4] The method of paragraph 1, wherein the promoter comprises orconsists of the polynucleotide sequence of SEQ ID NO: 1 or a subsequencethereof that has promoter activity.

[5] The method of paragraph 4, wherein the promoter comprises orconsists of the polynucleotide sequence of SEQ ID NO: 1.

[6] The method of paragraph 1, wherein the promoter is a hybrid promotercomprising one or more (e.g., several) portions of the polynucleotidesequences of SEQ ID NO: 1.

[7] The method of paragraph 1, wherein the promoter is a tandem promotercomprising one or more (e.g., several) polynucleotide sequences of SEQID NO: 1 or a subsequence thereof that retains promoter activity.

[8] The method of paragraph 7, wherein the promoter is a tandem promotercomprising one or more (e.g., several) polynucleotide sequences of SEQID NO: 1.

[9] The method of paragraph 7 or 8, wherein the tandem promotercomprises two or more (e.g., several) promoters.

[10] The method of paragraph 9, wherein the two or more (e.g., several)promoters of the tandem promoter simultaneously promote thetranscription of the polynucleotide.

[11] The method of paragraph 10, wherein one or more (e.g., several) ofthe two or more (e.g., several) promoters of the tandem promoter promotethe transcription of the polynucleotide encoding the polypeptide atdifferent stages of growth of the fungal host cell.

[12] The method of any of paragraphs 1-11, wherein the fungal host cellcontains one or more (e.g., several) copies of the polynucleotideencoding the polypeptide.

[13] The method of any of paragraphs 1-11, wherein the fungal host cellcontains one copy of the polynucleotide encoding the polypeptide.

[14] The method of any of paragraphs 1-13, wherein the polypeptide isselected from the group consisting of an antigen, enzyme, growth factor,hormone, immunodilator, neurotransmitter, receptor, reporter protein,structural protein, and transcription factor.

[15] The method of any of paragraphs 1-14, wherein the polypeptide isnative or foreign to the fungal host cell.

[16] The method of any of paragraphs 1-15, wherein the polynucleotide iscontained in the chromosome of the fungal host cell.

[17] The method of paragraph 1, wherein the polynucleotide is containedon an extrachromosomal element.

[18] The method of any of paragraphs 1-17, wherein the fungal host cellis a filamentous fungal cell.

[19] The method of any of paragraphs 1-17, wherein the fungal host cellis a yeast cell.

[20] An isolated promoter selected from the group consisting of (i) apromoter comprising a nucleotide sequence having at least 60% sequenceidentity to SEQ ID NO: 1; (ii) a promoter comprising a nucleotidesequence that hybridizes under at least medium stringency conditionswith SEQ ID NO: 1 or the full-length complement thereof; (iii) apromoter comprising SEQ ID NO: 1; (iv) a promoter comprising asubsequence of (i), (ii), or (iii) that retains promoter activity; and(v) a mutant, hybrid, or tandem promoter of (i), (ii), (iii), or (iv).

[21] The promoter of paragraph 20, which comprises a nucleotide sequencehaving at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 81%, at least 82%, at least 83%, at least 84%, at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%sequence identity to SEQ ID NO: 1.

[22] The promoter of paragraph 20, which comprises a nucleotide sequencethat hybridizes under medium stringency conditions, medium-highstringency conditions, high stringency conditions, or very highstringency conditions with SEQ ID NO: 1 or the full-length complementthereof.

[23] The promoter of paragraph 20, which comprises or consists of thepolynucleotide sequence of SEQ ID NO: 1 or a subsequence thereof thathas promoter activity.

[24] The promoter of paragraph 23, which comprises or consists of thepolynucleotide sequence of SEQ ID NO: 1.

[25] The promoter of paragraph 20, which is a hybrid promoter comprisingone or more (e.g., several) portions of the polynucleotide sequences ofSEQ ID NO: 1.

[26] The promoter of paragraph 20, which is a tandem promoter comprisingone or more (e.g., several) polynucleotide sequences of SEQ ID NO: 1 ora subsequence thereof that retains promoter activity.

[27] The promoter of paragraph 26, which is a tandem promoter comprisingone or more (e.g., several) polynucleotide sequences of SEQ ID NO: 1.

[28] The promoter of paragraph 26 or 27, wherein the tandem promotercomprises two or more (e.g., several) promoters.

[29] The promoter of paragraph 28, wherein the two or more (e.g.,several) promoters of the tandem promoter simultaneously promote thetranscription of a polynucleotide encoding a polypeptide.

[30] The promoter of paragraph 28, wherein one or more (e.g., several)of the two or more (e.g., several) promoters of the tandem promoterpromote the transcription of a polynucleotide encoding a polypeptide atdifferent stages of growth of the fungal host cell.

[31] A nucleic acid construct comprising a polynucleotide encoding apolypeptide operably linked to the promoter of any of paragraphs 20-30.

[32] A recombinant expression vector comprising the nucleic acidconstruct of paragraph 31.

[33] A recombinant host cell comprising the nucleic acid construct ofparagraph 31

[34] The recombinant host cell of paragraph 33, which is a filamentousfungal cell.

[35] The recombinant host cell of paragraph 33, which is a yeast cell.

[36] A nucleic acid construct comprising (a) a targeting sequence, (b)the promoter of any of paragraphs 20-30, (c) an exon, and (d) asplice-donor site.

[37] A nucleic acid construct comprising (a) a targeting sequence, (b) apromoter of any of paragraphs 20-30, (c) an exon, (d) a splice-donorsite, (e) an intron, and (f) a splice-acceptor site, wherein thetargeting sequence directs the integration of elements (a)-(f) such thatelements (b)-(f) are operably linked to an endogenous gene

[38] A method for producing a polypeptide comprising (a) cultivating ahomologously recombinant cell, having incorporated therein atranscription unit comprising a promoter of any of paragraphs 20-30, anexon, and/or a splice donor site operably linked to a second exon of anendogenous polynucleotide encoding the polypeptide, under conditionsconducive for production of the polypeptide, wherein the polynucleotideencoding the polypeptide is foreign to the promoter; and (b) recoveringthe polypeptide.

The invention described and claimed herein is not to be limited in scopeby the specific embodiments herein disclosed, since these embodimentsare intended as illustrations of several aspects of the invention. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

What is claimed is:
 1. A nucleic acid construct comprising apolynucleotide encoding a polypeptide operably linked to a promoterselected from the group consisting of (i) a promoter comprising anucleotide sequence that has at least 95% sequence identity to SEQ IDNO: 1; and (ii) a promoter comprising SEQ ID NO: 1; wherein thepolynucleotide encoding the polypeptide is foreign to the promoter. 2.The nucleic acid construct of claim 1, wherein the promoter comprises anucleotide sequence having at least 96% sequence identity to SEQ IDNO:
 1. 3. The nucleic acid construct of claim 1, wherein the promotercomprises a nucleotide sequence having at least 97% sequence identity toSEQ ID NO:
 1. 4. The nucleic acid construct of claim 1, wherein thepromoter comprises a nucleotide sequence having at least 98% sequenceidentity to SEQ ID NO:
 1. 5. The nucleic acid construct of claim 1,wherein the promoter comprises a nucleotide sequence having at least 99%sequence identity to SEQ ID NO:
 1. 6. The nucleic acid construct ofclaim 1, wherein the promoter comprises the polynucleotide sequence ofSEQ ID NO:
 1. 7. The nucleic acid construct of claim 1, wherein thepromoter sequence of SEQ ID NO: 1 is contained in a tandem promoter. 8.A recombinant host cell comprising the nucleic acid construct ofclaim
 1. 9. The recombinant host cell of claim 8, which is a filamentousfungal cell.
 10. The recombinant host cell of claim 8, which is a yeastcell.